Synthesis and NMR Studies of ¹³C-Labeled Vitamin D Metabolites¹

Abstract

Isotope-labeled drug molecules may be useful for probing by NMR spectroscopy the conformation of ligand associated with biological hosts such as membranes and proteins. Triple-labeled [7,9,19-(13)C(3)]-vitamin D(3) (56), its 25-hydroxylated and 1 alpha,25-dihydroxylated metabolites (58 and 68, respectively), and other labeled materials have been synthesized via coupling of [9-(13)C]-Grundmann's ketone 39 or its protected 25-hydroxy derivative 43 with labeled A ring enyne fragments 25 or 26. The labeled CD-ring fragment 39 was prepared by a sequence involving Grignard addition of [(13)C]-methylmagnesium iodide to Grundmann's enone 28, oxidative cleavage, functional group modifications leading to seco-iodide 38, and finally a kinetic enolate S(N)2 cycloalkylation. The C-7,19 double labeling of the A-ring enyne was achieved by the Corey-Fuchs/Wittig processes on keto aldehyde 11. By employing these labeled fragments in the Wilson-Mazur route, the C-7,9,19 triple-(13)C-labeled metabolites 56, 58, and 68 as well as other (13)C-labeled metabolites have been prepared. In an initial NMR investigation of one of the labeled metabolites prepared in this study, namely [7,9,19-(13)C(3)]-25-hydroxyvitamin D(3) (58), the three (13)C-labeled carbons of the otherwise water insoluble steroid could be clearly detected by (13)C NMR analysis at 0.1 mM in a mixture of CD(3)OD/D(2)O (60/40) or in aqueous dimethylcyclodextrin solution and at 2 mM in 20 mM sodium dodecyl sulfate (SDS) aqueous micellar solution. In the SDS micellar solution, a double half-filter NOESY experiment revealed that the distance between the H(19Z) and H(7) protons is significantly shorter than that of the corresponding distance calculated from the solid state (X-ray) structure of the free ligand. The NMR data in micelles reveals that 58 exists essentially completely in the alpha-conformer with the 3 beta-hydroxyl equatorially oriented, just as in the solid state. The shortened distance (H(19Z))-H(7)) in micellar solutions as compared to that in the solid state is most easily rationalized on the basis that the 5(10)-torsion angle in 58 is decreased in micellar solutions as compared to that in the solid state.

Identification of an alternative ligand-binding pocket in the nuclear vitamin D receptor and its functional importance in 1α,25(OH) 2 -vitamin D 3 signaling

“…3), but surprisingly, an Ϸ7 kcal͞mol lower A pocket I E compared with 1,25D [ Table 1; Ϫ84 (see §) vs. Ϫ91 kcal͞mol]. This energy loss may be compensated by a higher population of 25D molecules in the agonistic, ␣-chair, 6-s-trans conformation local to the hydrophobic membrane [25D favors ␣-chair 100% in SDS micelles (35)]. In addition, 25D has a much weaker G pocket I E (Table 1) than 1,25D; therefore, the difference in I E between the G and A pockets (⌬I E ) is low compared with JN and 1,25D ( Table 1), suggesting that it should show an increased selectivity for the A pocket.…”

“…2 A), whereas the dense bands in the 25(OH)D lane are c1 and the Ϸ30-kDa band (c3). Input represents 35 S-VDR not subjected to trypsin treatment. different ratio of ER␣ ensembles, provide a plausible explanation for EST's poor transactivation capability, yet potent bone fortification properties (11).…”

A perspective on how the Vitamin D sterol/Vitamin D receptor (VDR) conformational ensemble model can potentially be used to understand the structure–function results of A-ring modified Vitamin D sterols

“…Importantly HL shows a shift to the ␣-chair (70: 30) in acetone-d 6 (dielectric = 20.7), but it has been shown that the A-ring conformational profile for 1,25D remains the same in more polar solvents. On the other hand 25D shows a shift to the ␣-chair in both more polar (72%) and non-polar solvents (100% [66]). intrinsic conformational flexibility in their side-chain and seco-B-ring regions [13,34,35].…”

A Concise Catalytic Route to the Marine Sesquiterpenoids (–)‐Clavukerin A and (–)‐Isoclavukerin A

“…Much to our surprise, treatment of resultant ketone 6a with an excess amount of methylene(triphenyl)phosphorane in benzene at reflux not only caused methylenation of the ketone, but it also converted intermediate dibromoolefin 10a 16 into halogen‐free acetylene 3a . Although the mechanism of this reaction is not entirely clear,17,18 this one‐pot process directly provided us with the requisite substrate for the crucial domino metathesis. Whereas neither the Grubbs I nor the Grubbs II catalyst effected dienyne metathesis, application of phosphane‐free Hoveyda–Blechert catalyst 11 19 in refluxing toluene brought about the desired transformation of 3a under an ethylene atmosphere 20.…”